ORIGINAL ARTICLE
The phenolic contents, antioxidant and
anticholinesterase activity of section Amaracus
(Gled.) Vogel and Anatolicon Ietsw.
of Origanum L. species
Zu¨leyha O¨zer
a,*, Ahmet C. Go¨ren
b,c, Turgut K
ılıc¸
d, Merve O¨ncu¨
e, Sema C¸ar
ıkc¸ı
f,
Tuncay Dirmenci
ga
Medicinal and Aromatical Plants Programme, Altinoluk Vocational School, Balikesir University, 10870 Altinoluk, Edremit-Balikesir, Turkey
bBezmialem Vakıf University, Faculty of Pharmacy, Department of Analytical Chemistry, 34093 Fatih, Istanbul, Turkey c
Bezmialem Vakıf University, Drug Application and Research Center ( _ILMER), 34093 Istanbul, Turkey
dNecatibey Education Faculty, Department of Science Educations, Balikesir University, 10010 Balikesir, Turkey e
Faculty of Sciences and Arts, Department of Chemistry, Balikesir University, Campus of C¸ag˘ısß, Balikesir 10100, Turkey
f
Vocational School, Izmir Democracy University, 35330 Izmir, Turkey
g
Necatibey Education Faculty, Department of Biology Educations, Balikesir University, 10010 Balikesir, Turkey Received 20 November 2019; accepted 28 January 2020
Available online 6 February 2020
KEYWORDS Origanum; Amaracus; Anatolicon; Essential oil; Phenolics; Antioxidant activity; AChE; BChE
Abstract Origanum boissieriIetsw., O. saccatum P.H.Davis, O. solymicum P.H.Davis and O. ayli-niaeDirmenci & T.Yazıcı belonging to sect. Amaracus (Gled.) Vogel, O. sipyleum L. and O. hyper-icifolium O.Schwarz & P.H.Davis belonging to sect. Anatolicon Ietsw. were analyzed for their chemical composition of essential oil and phenolic components. The essential oil compositions were analysed by using GC-MS and GC-FID. The phenolic contents of the chloroform, acetone, and methanol extracts were analyzed using LC-MS/MS. Antioxidant activities of the extracts were investigated by using three methods; DPPH free radical scavenging activity,b-carotene linoleic acid assays and CUPRAC assays. The essential oil compositions of the section Amaracus were found to be as carvacrol type (O. ayliniae, O. boissieri) and p-cymene type (O. saccatum, O. solymicum). In the section of Anatolicon, while O. sipyleum was found as c-terpinene type, O. hypericifolium was carvacrol type. In the extracts, the most abundant components were determined as flavonoids, cou-maric acids and derivatives. Especially rosmarinic acid and penduletin were detected in high
* Corresponding author.
E-mail address:zuleyhaozer@balikesir.edu.tr(Z. O¨zer). Peer review under responsibility of King Saud University.
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https://doi.org/10.1016/j.arabjc.2020.01.025
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amounts. Among the studied species, extracts of O. ayliniae showed quite good activity for all meth-ods. The extracts from all species showed remarkable antioxidant activity. Inhibition capability of the extracts against acetyl and butyrylcholinesterase enzymes (AChE and BChE) were determined. The extracts were found as inactive against AChE. The moderate inhibition capacity observed against BChE.
Ó 2020 The Authors. Published by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
1. Introduction
Many species of aromatic plants have been used in the treat-ment of various diseases as spice among the population since ancient times. Especially in the last two decades, the members of Lamiaceae (Labiatae) family has become important (Baser, 1993; Celep and Dirmenci, 2017). Turkey is respected as an important gene-centre for the Lamiaceae family. The family is represented by 48 genera, 603 species and 782 taxa in Turkey (Celep and Dirmenci, 2017; Yılmaz et al., 2017). The rate of endemism in the family is 44% (Yılmaz et al., 2017).
One of the most used Lamiaceae members is Origanum L. The genus Origanum comprises 43 species in the world. The species are mainly concentrated in the temperate regions of the Mediterranean and South-West Asia. In Turkey, the genus Origanumconsist of 21 species (24 taxa), 13 of which are ende-mic and, 13 hybrids (12 of which are endeende-mic) (Dirmenci et al., 2018a; Dirmenci et al., 2018b; Dirmenci et al., 2019; Yılmaz et al., 2017). In Turkey, endemic species are concentrated within the Mediterranean region (Ietswaart, 1982). Sec-tion Amaracus (Gled.) Vogel consists of 4 endemic species: Ori-ganum boissieriIetsw. (Tasß mercan), O. saccatum P.H.Davis (Tahtacı kekig˘i), O. solymicum P.H.Davis (Kuz mercan) and a new species of O. ayliniae Dirmenci & T.Yazıcı. Section Ana-tolicon Ietsw. consists of 2 endemic species: O. sipyleum L. (Mor mercan) and O. hypericifolium O.Schwarz & P.H.Davis (Delik mercan). Origanum species have areas of usages in the pharmaceutical and food industry due to the antioxidant (Fotakis et al., 2016; Hajlaoui et al., 2016; Yan et al., 2016), antimicrobial (Hajlaoui et al., 2016), antibacterial (Evrendilek, 2015), cytotoxic (Sivropoulou et al., 1996), anti-fungal (Manohar et al., 2001), insecticidal (Pavela, 2004) and other biological activities of their essential oil, which rich in phenolic compounds, especially carvacrol.
Due to their biological activities (Yılmaz et al., 2017; Fotakis et al., 2016; Hajlaoui et al., 2016), many phytochemi-cal studies of Origanum species have been studied intensively (Yan et al., 2016; Evrendilek, 2015). The studies especially focused on essential oils and their biological activities. Pheno-lic compounds, especially carvacrol and thymol were deter-mined as the main compounds in the Origanum essential oils (Yılmaz et al., 2017; Sezik et al., 1993; Baser et al., 1993a; Baser et al., 1993b). In addition, there are many studies in the literature about phenolic composition of the extracts of Origanumspecies (Ozkan et al., 2007).
In the previous studies, O. solymicum (Tumen et al., 1994; Figue´re´do et al., 2006), O. saccatum (Tu¨men et al., 1995; Ozcan and Chalchat, 2009) and O. boissieri (Baser and Duman, 1998) were detected as p-cymene rich. O. hyperici-folium (Baser et al., 1994; Celik et al., 2010; Ili, 2016) and O. sipyleum (Baser et al., 1992) were found carvacrol,
p-cymene and c-terpinene rich. Biological activities of essential oil and extracts of the Origanum species have also been deter-mined such as antioxidant, antimicrobial, antibacterial, anti-fungal and antileishmanial activities (Baser et al., 1993a; Ozcan and Chalchat, 2009; Fakir et al., 2015; Dulger, 2006; Nakiboglu et al., 2007; Ozbilgin et al., 2014; Karago¨z et al., 2015). The studies especially focused on Anatolicon section. The antioxidant activity and total phenolic content of water, ethanol, methanol and acetone extracts (Nakiboglu et al., 2007), antioxidant, antimicrobial and free-radical-scavenging activities of the methanol extract and antileishmanical activity of O. sipyleum (Baser et al., 1993a; Ozbilgin et al., 2014; Karago¨z et al., 2015) were reported before. There are few stud-ies reporting the activity and phenolics of essential oil of O. hypericifolium and O. saccatum. Total phenolic content, antioxidant, antimicrobial activity (Celik et al., 2010), antifun-gal activity (Ocak et al., 2012) of O. hypericifolium, and antibacterial and antimicrobial activity of O. saccatum were reported in the literature (Ozcan and Chalchat, 2009; Sozmen et al., 2011).
There is only one study in the literature for Amaracus sec-tion. The antimicrobial activity of O. solymicum was investi-gated previously (Dulger, 2006). O. ayliniae is a newly identified species and has been reported to belong to the Amaracussection (Dirmenci et al., 2018a). Its chemical compo-nents and activities were studied for the first time in this study. The main reason for the unique activity of aromatic plants is the chemical components which they contain. Not only essential oils but also to determine other secondary metabolites is important. Origanum species, which are rich in essential oils, have been studied with many studies, but there are few studies about their phenolic contents (Yılmaz et al., 2017). The objec-tives of this study are to investigate antioxidant and anti-cholinesterase activities and to determine the phenolic composition of the extracts obtained from Origanum species belonging to sect. Amaracus (O. boissieri, O. saccatum, O. soly-micum, O. ayliniae) and sect. Anatolicon (O. sipyleum, O. hypericifolium). Furthermore, the composition of the essential oils depends on factors such as year, climate, solar angle, and collection region; the volatile oil compositions of the species of these two sections have been re-examined.
2. Materials and methods 2.1. Plant material
Localities, coordinates and collector numbers of the Origanum species are given inTable 1. The species were identified by Dr. Tuncay Dirmenci at Balıkesir University. Voucher specimens were deposited at the Herbarium of Faculty of Education, Balıkesir University, Balıkesir, Turkey.
2.2. Chemicals
Chloroform (Merck), acetone (Merck) and methanol (Merck) were used for the preparation of the extracts. The compounds were used as standards in LC-MS/MS analysis given in the supplementary material. Stock solutions were prepared as 10 mg/L in methanol. HPLC grade methanol was purchased from Merck (Darmstadt, Germany). Calibration solutions were prepared in methanol in a linear range. Dilutions were performed using automatic pipettes and glass volumetric flasks (A class), which were stored at 20 °C in glass containers. 100 mg/L curcumin solution was freshly prepared, from which 50 lL was used as an Internal Standard (IS) in all experiments (C¸arıkc¸ı et al., 2018; Sagir et al., 2017).
2.3. General
LC-MS/MS experiments were performed by a ZivakÒ HPLC and ZivakÒ Tandem Gold Triple quadrupole (Istanbul, Tur-key) mass spectrometry, equipped with a SynergyMax C18 col-umn (250 2 mm i.d., 5-lm particle size). The compounds used as standards in LC-MS/MS analyses were given in the supplementary material. For the antioxidant and anti-cholinesterase activity, the absorbance (UV and visible range 230 nm to 750 nm) was measured using a multiplate reader (Beckman Coulter DTX 880 Multimode Detector). GC-MS was conducted on Thermo Electron Trace 2000 GC model gas chromatography and Thermo Scientific TSQ GC-MS/ MS. A Phenomenex DB5 fused silica column (30 m 0.32 m m, with 0.25mm film thickness) was used with helium as a car-rier gas at 1 mL/min flow rate (138 kPa). The detailed proce-dures were given in thesupplementary material.
2.4. Essential oil
The aerial parts of Origanum species (100 g of each) which were air-dried in shade, were chopped into small pieces and subjected to hydrodistillation with water for 4 h, using a Clevenger-type apparatus to produce the essential oil. The obtained essential oils were stored in amber vials at 4°C for further analyses. Essential oil yields of species are 0.51%, 1.13%, 0.65%, 0.60%, 0.73% and 0.26% from O. boissieri,
O. saccatum, O. solymicum, O. ayliniae, O. sipyleum and O. hypericifolium,respectively.
2.5. Preparation of extracts
The air-dried grinded approximately 100 g of plant samples were directly extracted with methanol for 15 days. After filtra-tion and evaporafiltra-tion, they were named M1. Also, another 100 g of the plant was extracted with chloroform (C) for 15 days. After filtration and evaporation, the residue was extracted with acetone (Ac) and methanol (MeOH) for 15 days, respectively. They were named C, Ac, and M2. All the extracts were kept at 20 °C until they were used for experimental studies.
2.6. Determination of antioxidant activity 2.6.1.b-carotene bleaching method
The antioxidant activity was evaluated using b-carotene-linoleic acid model system (Miller, 1971; Yılmaz et al., 2017). b-carotene (0.5 mg) in 1 mL of chloroform was added to 25 lL of linoleic acid, and 200 mg of Tween 40 emulsifier mix-ture. After evaporation of chloroform under vacuum, 100 mL of distilled water saturated with oxygen, was through vigorous shaking. A mixture of four thousand microlitres was trans-ferred into different test tubes containing different concentra-tions of the sample (10, 25, 50 and 100 lg/mL). As soon as the emulsion was added to each tube, the zero time absorbance was measured at 470 nm using a spectrophotometer. The emul-sion system was incubated for 2 h at 50°C. A blank, devoid of b-carotene, was prepared for background subtraction. BHA, BHT and a-tocopherol were used as standard compounds. In the end, IC50values of all samples were calculated.
2.6.2. DPPH free radical scavenging method
The free radical scavenging activity of the extracts was deter-mined spectrophotometrically by the DPPH (1,1-diphenyl-2-picrylhydrazyl) assay (Blois, 1958; Reddy et al., 2015; Ertas et al., 2015; Sreedhar et al., 2016; Halfon et al., 2019). In its radical form, DPPH absorbs at 517 nm, but upon reduction by an antioxidant or a radical species its absorption decreases. Briefly, 0.1 mM solution of DPPH in methanol was prepared
Table 1 List of the Origanum species with locality, altitude and collection time.
Code Collector Number
Species Locality Altitude
(m)
Coordinates Year
OB TD 4285 Origanum boissieri
Mersin: Tarsus, Between C¸amlıyayla and Saimdibi, 15th km 1842 N37 22 824 E34 55 510
16.08.2014
OS TD 4296 Origanum saccatum
Alanya: Between Go¨kbel and C¸o¨kelek plateau, 8th km 1372 N36 62 604 E32 33 147
17.08.2014
OSL TD 4302 Origanum solymicum
Antalya: Kemer, Kesme bog˘azı, under P. brutia, calcareous rocks 104 N36 59 767 E30 49 907 18.08.2014 OA TD 4435 Origanum ayliniae
Aydın: Kusßadası, Dilek Peninsula NationalPark, rocky slopes 1195 N37 39 412 E27 08 575
30.07.2015
OSP TD 4308 Origanum sipyleum
Denizli: Between Serinhisar and Denizli, 5th km 1039 N37 61 968
E29 26 801
19.08.2014
OH TD 4315 Origanum hypericifolium
Denizli: Honaz, Honaz mountain, north slope, on the road of Arpacık plateau, under P.nigra
1268 N37 72 990 E29 26 676
and 160mL of this solution was added to 40 mL of sample solu-tions in methanol at different concentrasolu-tions (10, 25, 50 and 100 lg/mL). These tubes were left in the dark for 30 min. The measurements were made at 517 nm. BHA, BHT and a-tocopherol were used as standard compounds. The potentials of samples on DPPH were determined and compared to the standards. In the end, IC50values of all samples were
calcu-lated. The reduction in absorbance shows the DPPH free rad-ical scavenging of samples capability.
2.6.3. The CUPRAC method
The reducing capacities of extracts were evaluated using CUPRAC method (Apak et al., 2008; Apak, 2019). Briefly, 1 mM DMF, 10 mM CuCl2, 7.5 mM Neocuproine, 1 M NH4
-CH3COO (pH 7.0) solution, and distilled water were mixed in
volume ratio 1:1:1:0.6. After 180 ul of the mixture was dis-persed into the wells, 25mL diluted compounds (dilution ratio 1:20) in EtOH. The samples were kept for 30 min at 25°C. The absorbance was measured at 450 nm against a reagent blank. Ethanol was used as a negative control. Curcumin was used as a positive control.
2.6.4. Determination of the anticholinesterase activity
In vitro inhibition of AChE and BChE of the samples was assessed by the spectrophotometric method developed by Ell-man, Courtney, Andres and Featherston (Ellman et al., 1961; Yılmaz et al., 2016; Reddy et al., 2015). Activities of AChE and BChE were designated using 5,50-dithiobis (2-nitrobenzoic) acid (DTNB) (Ellman et al., 1961; Yilmaz et al., 2016). The test solutions and 150 mL of 100 mM sodium phosphate buffer (pH 8.0) were mixed with AChE or BChE enzymes solutions. The mixture waited at 25 °C for 15 min. Then, 0.5 mM DTNB was added. The reaction was then initi-ated by the addition of acetylthiocholine iodide (0.71 mM) or butyrylthiocholinechloride (0.2 mM). The activity was mea-sured at 412 nm. Methanol was used as a solvent to dissolve test compounds and the controls. Inhibition % of AChE or BChE was determined by a comparison of the rates of reaction of samples relative to blank sample (ethanol in phosphate buf-fer pH 8.0) using the formula; [(E-S)/E] 100 where E is the activity of enzyme without test sample, and S is the activity of enzyme with test sample. Galanthamine (4 mg/mL) was used as a positive control. All tests were conducted in triplicate.
2.7. Statistical analysis
Statistical analyses were used to evaluate antioxidant activity results by One-way ANOVA test. (GraphPad, Software 8.3.0). p < 0.05 was taken as the minimum level of significance.
3. Results and discussion 3.1. Essential oil
A total of 61 different compounds were identified, constituting 97.8–100.0% of the total oil. The components were classified into 6 classes based on their chemical structures: hydrocarbons and derivatives, monoterpene hydrocarbons, oxygenated
monoterpenes, sesquiterpene hydrocarbons, oxygenated sesquiterpenes, and phenolic compounds. Essential oil compo-sitions of the species are given inTable 2. O. boissieri and O. hypericifoliumwere found to be rich in oxygenated monoter-penes. The main compound of the essential oil of O. boissieri and O. hypericifolium was carvacrol (30.1%, 68.8%, respec-tively). Other main compounds were determined as p-cymene (29.8%) and cis-b-terpineol (10.2%) for O. boissieri, borneol (9.2%) and (Z)-caryophyllene (5.4%) for O. hypericifolium. In the previous study, O. boissieri was found to be rich in p-cymene (42.8%) and carvacrol (17.57%) (Baser and Duman, 1998). The chemical composition of the O. hypericifolium essential oil was investigated by several studies. For the essen-tial oil obtain, two different methods were used: steam distilla-tion (SD) and direct thermal desorpdistilla-tion (DTD). The determined major compounds were as follow: p-cymene and carvacrol (Celik et al., 2010), p-cymene (37.26%), thymol (11.86%) and borneol (10.26%) (Fakir et al., 2015), in the fruit and flower parts p-cymene (34.33%), carvacrol (21.76%) and thymol (19.54%) (Ocak et al., 2012). In the study, which aimed to determine of the difference in the oil composition of devel-opment stages of the O. hypericifolium, carvacrol (64.33%) found to be the major component of the oil when collected before flowering, whereas p-cymene (36.10–47.75%) was the major component when collected while in full flowering (Baser et al., 1994). Unlike these studies, it was reported that thymol (59.3%) was found to the main compound of O. hyper-icifolium(Figue´re´do et al., 2006). O. ayliniae was dedected as oxygenated monoterpene rich and main compound was car-vacrol (53.7%), with carcar-vacrol methyl ether (14.4%) and p-cymene (13.9%). This is the first study of the essential oil com-position of O. ayliniae. O. saccatum, O. solymicum and O. sipy-leum were detected as monoterpene hydrocarbons rich. p-Cymene (37.9%, 29.6%, respectively), carvacrol (21.6%, 15.2%, respectively) and c-terpinene (12.5%, 12.7%, respec-tively) were the main compounds of the essential oils of O. sac-catumand O. solymicum. In different studies, essential oil of O. saccatum was characterized by its high content of p-cymene (Tu¨men et al., 1995; Ozcan and Chalchat, 2009; Sozmen et al., 2011). In the oil of O. solymicum, the major constituent was indentified as p-cymene (53.07%) (Tumen et al., 1994). O. sipyleum was found monoterpene hydrocarbon rich and the main compounds were detected as c-terpinene (28.7%), p-cymene (21.6%) and carvacrol (21.2%). In the previous study, the oils of O. sipyleum collected from four different locations were investigated and, c-terpinene (10.80–26.60%), p-cymene (3.76–36.60%), thymol methylether (trace-19.90%), carvacrol methylether (0.41–10.20%), thymol (0.23–7.30%) and car-vacrol (0.82–12.20%) were determined as main compounds (Baser et al., 1992).
In the present study, the essential oil composition of Amaracus and Anatolicon section has been analyzed to have different chemotypes. This study demonstrated the presence of O. boissieri, O. ayliniae and O. hypericifolium in carvacrol type, which is known for its antioxidant, antimicrobial (Mathela et al., 2010), antifungal (Vinciguerra et al., 2019) and acaricidal (Cetin et al., 2010) activities. O. saccatum and O. solymicumwere reported as p-cymene type, which is known for its antioxidant (Oliveira et al., 2015), acetylcholinesterase activity (Miyazawa and Yamafuji 2006) and antifungal (Kordali et al., 2008; Mirzania et al., 2018), phytotoxic and
Table 2 Essential oil composition of section Amaracus and Anatolicon.
Amaracus Anatolicon
No Compounds KI* OB** OS** OSL** OA** OSP** OH**
Hydrocarbons and derivatives
1 3-methyl nonane 971 – 0.1 3.6 – 0.6 t 2 1-octen-3-ol 979 1.4 0.5 – 0.4 1.4 3 3-octanol 991 – 0.1 0.2 – 0.1 0.1 4 2-methyl decane 1063 – 0.5 – 0.2 0.6 5 undecane 1100 – – 8.0 – 0.3 0.1 % identified – 1.6 12.8 – 1.6 2.2 Monoterpene hydrocarbons 6 a-thujene 930 – – 0.4 – 0.7 – 7 a-pinene 939 – 0.1 1.5 – 0.3 – 8 camphene 954 0.3 – 1.1 – t – 9 sabinene 975 – – 0.5 – – – 10 b-pinene 979 – 0.2 0.6 – 3.3 – 11 a-phellandrene 1003 – t 0.1 – 0.1 – 12 a-terpinene 1017 – 0.4 1.2 – 1.6 t 13 p-cymene 1025 29.8 37.9 29.6 13.9 21.6 1.6 14 limonene 1029 – 0.1 0.6 – 0.5 t 15 (E)-b -ocimene 1050 1.7 – – 1.7 0.4 – 16 c-terpinene 1060 0.2 12.5 12.7 – 28.7 1.3 % identified 32.0 51.2 48.3 15.6 57.2 2.9 Oxygenated monoterpenes 17 sabinene hydrate-cis 1070 0.1 – – 0.8 – – 18 a-terpinolene 1089 – – 0.3 – 0.1 0.1 19 pinene hydrate 1123 0.6 – – – – – 20 terpineol 1134 0.9 – – – – – 21 cis-b-terpineol 1144 10.2 – – – – – 22 camphor 1146 6.4 – 0.2 0.5 – – 23 menth-3-en-8-ol 1150 2.6 – – – – – 24 menthone 1153 0.4 – – 0.1 – – 25 trans-b-terpineol 1163 0.5 – – 0.1 – – 26 borneol 1169 – 1.8 9.0 – 1.8 9.2 27 4-terpineol 1177 – 2.2 0.8 – 0.6 1.3 28 a-terpineol 1189 – 5.4 1.2 – 0.3 1.0 29 myrtenol 1196 – 0.3 0.2 – – 0.1 30 carveol-cis 1229 – – – 0.7 – – 31 carvone 1243 – 1.8 – – 2.5 –
32 carvacrol methyl ether 1245 2.6 – – 14.4 – –
33 bornyl acetate 1289 – 0.2 0.3 – – –
34 thymol 1290 2.5 – – – – –
35 cymen-7-ol 1291 – 2.6 – – – 0.3
36 terpinene-7-al 1291 0.6 – – – – –
37 carvacrol, ethyl ether 1298 0.6 – – – – –
38 carvacrol 1299 30.1 21.6 15.2 53.7 21.2 68.8 % identified 58.1 35.9 28.4 70.3 26.5 80.8 Sesquiterpene hydrocarbons 37 d-elemene 1338 2.1 – – 3.1 – – 38 a-cubebene 1351 0.2 – – – – – 39 a-ylangene 1375 – – – 0.2 – – 40 a-copaene 1377 – – – – 1.1 – 41 b-bourbonene 1388 0.4 – – – – 0.2 42 b-elemene 1391 – 0.2 – – – – 43 (Z)-caryophyllene 1409 – 4.3 3.9 – 2.5 5.4 44 a-gurjunene 1410 – 0.1 – – – – 45 aromadendrene 1441 2.0 – – 5.6 – – 46 a-humulene 1455 0.2 0.7 0.3 – 0.3 0.3 47 E-b-farnesene 1457 0.2 0.1 – – – – 48 allo-aromadendrene 1460 1.0 – – 2.1 – – 49 s-muurolene 1480 – – 1.3 – 1.9 2.4 50 germacrene D 1485 – – 1.3 – 4.0 0.4 51 a-cadinene 1539 – – 0.1 – 0.8 0.2
insecticidal properties (Kordali et al., 2008). O. O. sipyleum was reported as c-terpinene type, which is known for its acetyl-cholinesterase (Miyazawa and Yamafuji 2006) and acaricidal (Cetin et al., 2010) activities.
3.2. Phenolic contents
The phenolic contents were analyzed under four groups; flavo-noids and derivatives, coumaric acid and derivatives, simple phenolics and others and dicarboxylic acid (Fig. 2). Identified compounds and their quantities are given inTables 3–6.
While the main phenolic components of the methanol extracts (M1 and M2) and the acetone extracts of species were shown differ in chemical structure, C extracts were determined rich in flavonoids and derivatives. Rosmarinic acid, pend-uletin, salvigenin, fumaric acid, kaempferol, gallic acid and pryrogallol are analyzed as most common compounds in the extracts. Main compounds of C extracts were analyzed as pen-duletin and salvigenin. Methanol extracts (M1 and M2) and Ac extracts were rich in rosmarinic acid. M2 extracts of O. boissieri, O. solymicum and O. sipyleum, Ac extracts of O. sac-catum, O. ayliniae and O. hypericifolium were determined as rich in phenolic compounds. Especially Ac extract of O. sacca-tumwas the richest (4092.48 mg/kg). O. saccatum and O. soly-micumwere found as the richest species, whereas O. ayliniae was the poorest in terms of phenolic compounds.
Rosmarinic acid was determined as the main components of most of the extracts: M1, Ac and M2 extracts of O. boissieri; M1, C and M2 extracts of O. saccatum; M1 and Ac extracts of O. solymicum; Ac and M2 extracts of O. sipyleum. There are few studies reporting the activity and phenolics of various extracts of O. sipyleum.Ozkan et al. (2007)reported the pres-ence of apigenin, carvacrol, hesperidin, naringenin, rutin and vitexin in O. sipyleum methanol extract. Total phenolic con-tents, DPPH, OH radicals scavenging and total antioxidant capacities of O. sipyleum were investigated (Nakiboglu et al., 2007). Total phenolic content, antioxidan activity of water, methanol and chloroform extracts of O. sipyleum, O. hyperici-folium, O. majoranaand O. onites were reported in the litera-ture (Semiz et al., 2018). Also,Zengin et al. (2019)reported that O. sipyleum can be considered as a good source of
pheno-lic compounds such as rosmarinic acid and phlorizin. The C extracts of O. boissieri, O. saccatum and O. hypericifolium, Ac and M2 extracts of O. ayliniae were found to be rich in a flavonoid derivatives penduletin. Another flavonoid deriva-tives salvigenin was determined as the main compound for the C extracts of O. solymicum, O. ayliniae and O. sipyleum, kaempferol was determined as a major compound for Ac extracts of O. saccatum and O. sipyleum. While fumaric acid was determined as the main compounds for M1 extracts of O. sipyleumand O. hypericifolium, gallic acid was determined in M2 extract of O. solymicum.
3.3. Activity
The antioxidant activities were determined mainly using three methods; DPPH free radical scavenging activity, b-carotene linoleic acid assays and CUPRAC assays. In the DPPH and b-carotene linoleic acid assays, the activities were determined at four concentrations: 10, 25, 50 and 100 lg/mL. BHA, BHT and a-tocopherol were used as standards. The results are given as 50% inhibition concentrations (IC50) inTable 7.
In the CUPRAC, TEACCUPRAC values of the extracts were
calculated by using curcumin as a reference. TEACCUPRAC
of curcumin was found as 0.9 mmol TR g 1(Fig. 1).
To evaluate the free radical scavenging effectiveness of extracts of species, DPPH method was used. Methanol (M1 and M2) and acetone (Ac) extracts of both section have good antioxidant activity for all tested methods. As shown in Table 7, among the studied species, all of the extracts of O. ayliniae exhibited a significant activity for b-carotene and DPPH methods when compared to that of standard antioxi-dants. In particular, O. ayliniae M1 extract exhibited a remark-able DPPH free radical scavenging activity. IC50values for the
radical scavenging activity of O. ayliniae M1 extract were found to be 7.63 lg/mL. Also, free radical scavenging activity of O. ayliniae extracts were compared to those of BHA, BHT and a-tocopherol. On the other hand, IC50values for BHA,
BHT and a-tocopherol were found to be 9.53 lg/mL, 11.04 lg/mL, 12.50 lg/mL, respectively. These results indi-cated that the free radical scavenging effect of O. ayliniae M1 extract was higher than those of BHA, BHT and Table 2 (continued)
Amaracus Anatolicon
No Compounds KI* OB** OS** OSL** OA** OSP** OH**
% identified 6.1 5.4 6.9 11.0 10.6 8.9 Oxygenated sesquiterpenes 52 spathulenol 1578 3.4 – 1.3 2.5 1.8 1.2 53 caryophyllene oxide 1583 0.1 3.0 2.1 0.1 0.5 1.7 54 a-cadinol 1654 0.3 – – – – – 55 ledol 1590 – 0.2 – – – 0.1 56 viridiflorol 1593 – – – – 0.2 t 57 a-cadinol 1660 – 0.1 – – – 0.1 58 valeranone 1675 – 0.3 0.2 – 0.2 0.1 59 a-bisabolol 1686 – 0.1 t – 0.2 – % identified 3.8 3.7 3.6 2.6 2.9 3.2 Total (%) 100.0 97.8 100.0 99.5 98.8 98.0 * KI Kovats indices.
Table 3 Phenolic contents of the M1 extracts.
Amaracus Anatolicon
OB* OS* OSL* OA* OSP* OH*
Flavonoids and derivatives
Kaempferol 7.91 ± 0.56 119.09 ± 8.41 65.44 ± 4.62 14.9 ± 1.05 150.61 ± 10.6 208.75 ± 14.73 Salvigenin – 104.83 ± 7.13 16.64 ± 1.13 97.06 ± 6.61 47.36 ± 3.22 29.11 ± 1.98 Penduletin 22.11 ± 2.24 169.52 ± 17.19 3.5 ± 0.36 103.77 ± 10.5 – 273.75 ± 27.75 Isorhamnetin – 41.59 ± 3.67 – – – – Quercetin – 13.47 ± 1.79 12.59 ± 1.67 – – 11.88 ± 1.58 Quercetagetin-3,6-dimethylether 14.33 ± 2.68 52.54 ± 9.84 1.63 ± 0.3 – – 3.19 ± 0.6 Quercitrin – – – – 22.91 ± 1.46 – Luteolin – 12.27 ± 3.15 5.02 ± 1.29 – 19.74 ± 5.07 40.26 ± 10.34 Luteolin-7-O-glucoside – – 9.6 ± 0.98 – 21.63 ± 2.2 – Luteolin-5-O-glucoside – – – – 134.15 ± 8.63 – Rutin 2.54 ± 0.17 15.02 ± 0.98 10.35 ± 0.68 – 1.79 ± 0.12 5.28 ± 0.35 Pelargonin – – – 50.65 ± 5.15 – –
Total (mg/kg dried herba) 46.89 528.33 124.77 266.38 398.19 572.22 Coumaric acids and derivatives
Caffeic acid 71.62 ± 14.17 78.98 ± 15.63 99.45 ± 19.68 – 112.33 ± 22.2 79.36 ± 15.71 (E)-Ferulic acid 70.18 ± 4.9 158.83 ± 11.1 143.73 ± 10.04 187.43 ± 13.1 130.43 ± 9.11 123.15 ± 8.61 Chlorogenic acid 13.09 ± 1.81 123.39 ± 17.09 70.07 ± 9.7 166.29 ± 23.0 11.12 ± 1.54 15.79 ± 2.19 Rosmarinic acid 1358.25 ± 104.15 1462.53 ± 112.1 2020.01 ± 154.8 – – –
Syringic acid 25.19 ± 1.7 – – – – –
Total (mg/kg dried herba) 1538.33 1823.73 2333.26 353.72 253.88 218.3 Simple phenolics and others
Gallic acid 5.67 ± 0.39 7.24 ± 0.5 7.79 ± 0.54 – 5.15 ± 0.36 5.81 ± 0.4 Pyrogallol – 29.95 ± 1.99 31.21 ± 2.08 690.73 ± 45.9 12.71 ± 0.85 27.5 ± 1.83 Total (mg/kg dried herba) 5.67 37.19 39 690.73 17.86 33.31 Dicarboxylic acid
Fumaric acid 34.58 ± 2.4 189.08 ± 13.11 201.59 ± 13.98 – 216.91 ± 15.0 258.25 ± 17.91 Total (mg/kg dried herba) 34.58 ± 2.4 189.08 ± 13.11 201.59 ± 13.98 – 216.91 ± 15.0 258.25 ± 17.91
1625.47 2578.33 2698.62 1310.83 886.84 1082.08
*
OB: O. boissieri, OS: O. saccatum, OSL: O. solymicum, OA: O. ayliniae, OSP: O. sipyleum, OH: O. hypericifolium.
Table 4 Phenolic content of the C extracts.
Amaracus Anatolicon
OB* OS* OSL* OA* OSP*
Flavonoids and derivatives
Kaempferol – 15.58 ± 1.1 – – – Salvigenin 25.28 ± 1.72 240.62 ± 16.38 17.03 ± 1.16 398.51 ± 27.12 18.79 ± 1.28 Penduletin 62.73 ± 6.36 418.7 ± 42.45 9.94 ± 1.01 206.54 ± 20.94 12.48 ± 1.27 Isorhamnetin – 85.98 ± 7.59 – 56.37 ± 4.11 – Quercetin – – – 10.25 ± 1.02 – Quercetagetin-3,6-dimethylether 35.53 ± 6.65 94.26 ± 17.65 – 26.33 ± 5.22 – Total (mg/kg dried herba) 123.54 855.14 26.97 698.00 31.27 Coumaric acids and derivatives
Caffeic acid 9.27 ± 1.83 8.41 ± 1.66 5.95 ± 1.18 – 5.71 ± 1.13 Chlorogenic acid 6.8 ± 0.94 7.78 ± 1.08 6.79 ± 0.94 – 8.00 ± 1.11
Syringic acid 25.19 ± 1.7 – – – –
Rosmarinic acid 3.72 ± 0.29 4.16 ± 0.32 4.7 ± 0.36 – 3.62 ± 0.28 Total (mg/kg dried herba) 44.98 20.35 17.44 – 17.33 Simple phenolics and others
Gallic acid 5.67 ± 0.39 – – – –
Total(mg/kg dried herba) 5.67 – – – –
Dicarboxylic acid
Fumaric acid 34.58 ± 2.4 – – – –
Total (mg/kg dried herba) 34.58 ± 2.4 – – – –
208.77 875.49 44.41 698.00 48.60
a-tocopherol. Lower IC50value indicates higher radical
scav-enging activity. O ayliniae M1 extract consisted of pyrogallol, ferulic acid and chlorogenic acid as dominant compounds. According to recent reports, pyrogallol showed effective radi-cal scavenger activity (Ozturk Sarikaya, 2015). In general, free radical scavenging and antioxidant activities of the phenolic compounds depend on the number of hydroxyl groups (– OH) and their positions on the aromatic rings (Ahmad et al., 2018; Tian and Liu, 2018; Phương et al., 2018; Lan et al., 2018). The Chloroform (C) extracts of O. boissieri, O. sacca-tum, O. solymicum, O. sipyleum and O. hypericifolium were showed lowest activities. IC50 values for DPPH free radical
scavenging activities for O. boissieri, O. saccatum, O. solymi-cum, O. sipyleumand O. hypericifolium C extracts were found to be 91.62 lg/mL, 91.76 lg/mL, 96.92 lg/mL, 95.03 lg/mL and 94.30 lg/mL, respectively. It could be concluded that weak activity observed in that species is associated with a low amount of phenolic compounds.
O. ayliniaeM1 and M2 extracts showed great lipid peroxi-dation inhibition in theb-carotene-linoleic acid system (IC50
7.95 lg/mL, 7.99 lg/mL, respectively). IC50 values of BHA
and BHT were found to be 6.12 lg/mL; 6.35 lg/mL and
6.13 lg/mL; 6.47 lg/mL, respectively. None of the tested extracts showed greater antioxidant activity than BHA or BHT. On the other hand, IC50values for a-tocopherol were
found to be 9.47 lg/mL and 9.11 lg/mL. The results show that O. ayliniaeM1 and M2 extracts exhibited higher activities than the a-tocopherol. The lower inhibition value was found in C extracts.
Cu2+ reducing ability (CUPRAC method) is frequently used to determine the reducing powers of curcumin and M1, C, Ac and M2 extracts of Origanum species (Fig. 1). In CUPRAC method, same as other methods, O. ayliniae extracts have better activity than the other studied species as well as curcumin, which was used as a standard compound. Cu2+ reducing powers of the O. ayliniae extracts decreased as fol-lows: M1 (2.74 mmol TR g 1), M2 (2.62 mmol TR g 1), Ac (1.59 mmol TR g 1), C (1.27 mmol TR g 1) and curcumin
(0.9 mmol TR g 1). Additionally, rosmarinic acid-rich extracts M1 and M2 of the species had the best activity.
Acetylcholinesterase (AChE) enzyme plays an important role of the cholinergic system in the central and peripheral ner-vous system (Gu¨lc¸in et al., 2019). Acetylcholine (ACh) as a neu-rotransmitter decreases due to the decline in acetyltransferase
Table 5 Phenolic contents of the Ac extracts.
Amaracus Anatolicon
OB* OS* OSL* OA* OSP* OH*
Flavonoids and derivatives
Kaempferol 49.68 ± 3.51 1307.04 ± 92.25 420.71 ± 29.69 10.59 ± 0.75 646.58 ± 45.64 648.93 ± 45.8 Kaempferol-3-rutinoside – 21.73 ± 1.96 3.66 ± 0.33 – 2.51 ± 0.23 8.3 ± 0.75 Salvigenin – 173.9 ± 11.83 45.26 ± 3.08 246.39 ± 16.7 – – Penduletin 17.15 ± 1.74 378.86 ± 38.41 15.53 ± 1.57 403.86 ± 40.9 20.33 ± 2.06 478.88 ± 48.5 Isorhamnetin 105.85 ± 9.34 10.79 ± 0.95 – – – Quercetin 27.84 ± 3.7 281.61 ± 37.44 173.82 ± 23.11 – – 135.46 ± 18.0 Quercetagetin-3,6-dimethylether 16.74 ± 3.14 81.92 ± 15.34 14,63 ± 2.74 58.36 ± 8.59 – 5.63 ± 1.05 Isoquercetin – – 1.91 ± 0.55 – – – Luteolin 2.65 ± 0.68 337.74 ± 86.75 94.86 ± 24.37 3.58 ± 0.15 180.73 ± 46.42 185.34 ± 47.6 Luteolin-7-O-glucoside – 1.7 ± 0.17 7.19 ± 0.73 – 6.68 ± 0.68 – Apigenin – – – 10.38 ± 0.89 – – Rutin 1.49 ± 0.1 114.44 ± 7.5 2.21 ± 0.14 – 2.00 ± 0.13 2.33 ± 0.15 Pelargonin – – – 88.59 ± 9.02 – –
Total (mg/kg dried herba) 115.55 2804.79 790.57 821.75 858.83 1464.87 Coumaric acids and derivatives
p-Coumaric acid – 5.97 ± 0.92 – – 4.84 ± 0.74 –
Caffeic acid 46.78 ± 9.26 77.79 ± 15.39 73.4 ± 14.53 – 100.8 ± 19.95 27.8 ± 5.5 (E)-Ferulic acid – 2.82 ± 0.2 3.28 ± 0.23 – 8.56 ± 0.6 –
Chlorogenic acid 6.91 ± 0.96 – 7.45 ± 1.03 – – 6.84 ± 0.95 Rosmarinic acid 403.00 ± 30.9 1295.34 ± 99.33 967.42 ± 74.18 – 1760.8 ± 135.0 175.84 ± 13.5 Total (mg/kg dried herba) 456.69 1381.92 1051.55 – 1875.00 210.48 Simple phenolics and others
Gallic acid 6.05 ± 0.42 7.54 ± 0.52 7.95 ± 0.55 – 7.42 ± 0.51 5.1 ± 0.35
Ellagic acid 7.91 ± 0.53 – – – – 14.37 ± 0.96
Vanillin – – – – 7.87 ± 0.72 –
Total (mg/kg dried herba) 13,96 7,54 7.95 – 15,29 19.47 Dicarboxylic acid Fumaric acid 27.09 ± 1.88 4.08 ± 0.28 – – 59.91 ± 4.15 – Total (mg/kg dried herba) 27.09 ± 1.88 4.08 ± 0.28 – – 59.91 ± 4.15 – 613.29 4092.48 1850.07 821.75 2809.00 1694.82 *
activity and choline (Ch). It was reported that the reduction of ACh and BCh levels in hippocampus and cortex in the brain is the most remarkable biochemical change in Alzhemer Diease (AD) patients. As a result of this, one of the treatment approaches for AD is inhibition of AChE and BChE enzymes that break down ACh and BCh (Gu¨lc¸in et al., 2019; Taslimi et al., 2020). Inhibitors of AChE, such as galanthamine, are
used frequently to treat the symptoms of AD (Loizzo et al., 2009), which hydrolyses the acetylcholine compound involved in the communication between synapses in the nervous system. The less specific BChE has recently been a focus of research, because BChE concentration stays the same, or is even up-regulated, while AChE is dramatically down-regulated in the brains of patients suffering from AD. AChE inhibitors had a
Table 6 Phenolic contents of the M2 extracts.
Amaracus Anatolicon
OB* OS* OSL* OA* OSP* OH*
Flavonoids and derivatives
Kaempferol 56.5 ± 3.99 99.48 ± 7.02 685.87 ± 48.41 8.56 ± 0.6 114.06 ± 8.05 – Salvigenin – – – 158.31 ± 10.77 – – Penduletin – 14.37 ± 1.46 – 239.61 ± 24.29 – – Quercetin 8.95 ± 1.19 4.66 ± 0.62 – – 24.41 ± 1.56 – Quercetagetin-3,6-dimethylether – 5.16 ± 0.97 – – – – Luteolin 5.55 ± 1.42 16.09 ± 4.13 – – 30.23 ± 7.76 – Luteolin-7-O-glucoside – 4.27 ± 0.43 – – 62.88 ± 6.4 – Luteolin-5-O-glucoside – – – – 36.92 ± 2.38 – Rutin 6.22 ± 0.41 142.22 ± 9.32 – – – – Pelargonin – – – 53.6 ± 5.45 – –
Total (mg/kg dried herba) 77.22 286.25 685.87 460.08 268.50 – Coumaric acids and derivatives
Caffeic acid 218.59 ± 43.26 122.23 ± 24.19 – – 135.21 ± 26.76 – (E)-Ferulic acid 182.95 ± 12.78 210.18 ± 14.69 – 99.69 ± 6.97 136.57 ± 9.54 – Chlorogenic acid 10.83 ± 1.5 224.61 ± 31.1 437.23 ± 60.55 50.17 ± 6.95 9.23 ± 1.28 – Rosmarinic acid 2085.33 ± 159 2421.83 ± 185 – – 2495.11 ± 191.32 –
Syringic acid 222.3 ± 14.97 – – – – –
Total (mg/kg dried herba) 2720.00 2978.85 437.23 149.86 2776.12 – Simple phenolics and others
Gallic acid – 7.02 ± 0.49 1046.31 ± 72.5 5.67 ± 0.39 5.12 ± 0.36 – Pyrogallol 20.23 ± 1.35 17.23 ± 1.15 – – 13.62 ± 0.91 – Total (mg/kg dried herba) 20.23 24.25 1046.31 5.67 18.74
Dicarboxylic acid
Fumaric acid 218.87 ± 15.1 268.38 ± 18.61 – – 216.14 ± 14.99 – Total (mg/kg dried herba) 218.87 ± 15.1 268.38 ± 18.61 – – 216.14 ± 14.99 – 3036.32 3557.73 2169.41 615.61 3044.62 –
*
OB: O. boissieri, OS: O. saccatum, OSL: O. solymicum, OA: O. ayliniae, OSP: O. sipyleum, OH: O. hypericifolium.
Table 7 DPPH free radical scavenging activity and lipid peroxidation of the extracts, BHA, BHT and a-tocopherol (IC50mg/mL).
M1 C Ac M2
b-carotene DPPH b-carotene DPPH b-carotene DPPH b-carotene DPPH OB* 31.23 ± 3.01 91.99 ± 6.78 91.66 ± 3.00 91.62 ± 13.27 40.58 ± 17.95 84.79 ± 1.30 12.64 ± 1.04 29.83 ± 1.10 OS* 9.72 ± 0.53 32.64 ± 5.46 91.37 ± 8.69 91.76 ± 1.60 15.39 ± 4.81 47.27 ± 11.04 12.33 ± 0.83 20.11 ± 2.49 OSL* 9.19 ± 0.46 27.53 ± 4.49 89.12 ± 0.82 96.92 ± 2.11 10.19 ± 1.01 42.22 ± 2.31 8.68 ± 0.27 11.64 ± 0.49 OA* 7.95 ± 7.95 7.63 ± 0.17 13.61 ± 3.35 38.86 ± 0.80 9.64 ± 0.76 29.58 ± 3.13 7.99 ± 0.97 9.59 ± 0.67 OSP* 13.59 ± 3.98 20.12 ± 4.39 88.05 ± 7.73 95.03 ± 2.95 35.19 ± 5.75 42.39 ± 3.36 8.38 ± 0.31 18.99 ± 0.50 OH* 9.13 ± 1.41 30.45 ± 0.54 89.56 ± 2.65 94.30 ± 2.82 18.83 ± 1.92 38.29 ± 2.03 9.01 ± 0.22 31.32 ± 2.77 BHA 6.12 ± 0.07 10.14 ± 0.81 6.13 ± 0.08 11.86 ± 0.20 6.14 ± 0.05 9.59 ± 0.66 6.13 ± 0.10 9.53 ± 0.30 BHT 6.35 ± 0.29 11.42 ± 2.49 6.33 ± 0.09 11.05 ± 0.75 6.39 ± 0.07 11.57 ± 1.63 6.47 ± 0.13 11.04 ± 0.18 a-Tocopherol 9.47 ± 1.78 12.93 ± 2.99 9.29 ± 0.09 12.44 ± 0.65 9.27 ± 0.96 12.56 ± 0.71 9.11 ± 0.21 12.50 ± 0.08
IC50values are mean ± SD (n = 3). *
common usage in medicine, especially for the treatment of AD. They have been used in clinical trials, including natural sub-stances. Phenolic compounds had been also recognized as AChE inhibitors and promising lead compounds for AD. Therefore, finding new AChE and BChE sources is very impor-tant and one of the best sources is plants.
Anticholinesterase activities of extracts of species were determined at 200 mg/mL and galanthamine was used as a standard compound. The results are given in theTable 8.
The M1 and Ac extracts of O. hypericifolium showed mod-erate AChE (51.32 ± 2.69% and 49.80 ± 0.53%, respectively) and BChE (54.91 ± 0.85% and 62.80 ± 0.55%, respectively) inhibitory activity. In contrast, the M1, C and Ac extracts of O. boissieri, O. saccatum, O. solymicumand O. sipyleum was only exhibited activity against butyrylcholinesterase enzyme. M2 extract of O. solymicum showed mild butyrylcholinesterase inhibitory activity (8.55 ± 0.40%), while M2 extracts of other species exhibited no activity.Orhan et al., (2007)reported that there was no correlation between acetylcholinesterase and butyrylcholinesterase enzyme inhibition and phenolic contents. They had reported that some of these compounds are not inhi-bitor for AChE and BChE. Rather than phenolic acids, flavo-noid derivatives such as quercetin, genistein, luteolin-7-O-rutinoside were found to be more effective inhibitors (Orhan et al., 2009; Jung and Park, 2007). Structural requirements of flavonoids as AChE and BChE inhibitors have been investi-gated (Orhan et al., 2007; Panche et al., 2016). It was reported that catechol moiety on ring B and this moiety has positive effects on the enzyme-inhibiting activities of quercetin con-tributing to its binding with the enzyme (Orhan et al., 2007; Orhan et al., 2009). Amongst the tested extracts, the Ac and direct methanol (M1) extracts of O. hypericifolium were shown to have the best acetylcholinesterase (54.91 ± 0.85%) and butyrylcholinesterase (62.08 ± 0.55%) inhibitory activity, which might be due to high flavonoid content of M1 and Ac extracts of O. hypericifolium. These results are consistent with the literature.
4. Conclusion
All member of the section of Amaracus and Anatolicon of Ori-ganumare endemic species for Turkey. Also, O. ayliniae was just identified recently and added to sect. Amaracus. In the pre-sent study, espre-sential oil and phenolic composition of the methanol, chloroform and acetone extracts of both sections
were investigated. Also, the antioxidant and anticholinesterase activities of the extracts were determined. There are numerous reports on the chemical composition of essential oil of the spe-cies. In our study, it was found that the oil composition of the sect. Amaracus and sect. Anatolicon were different chemotypes, and also, our findings are consistent with the literature. It can be said that the differences in the chemical composition of essential oils depend on climatic, geographic conditions, har-vest period, distillation time and distillation technique. To the best of our knowledge, this is the first report on the pheno-lic composition and anticholinesterase activities of the species. A considerable qualitative and quantitative variation was observed in the phenolic compounds of extracts of the species. Rosmarinic acid, penduletin, salvigenin, fumaric acid, kaemp-ferol, gallic acid and pryrogallol were determined as the main phenolic compounds of the species. The richest extracts in
Fig. 2 Standards chromatogram of secondary metabolites (Phenolics and Others) by LC-MS/MS (5 mg/L).
0 0.5 1 1.5 2 2.5 3 mm ol T R g -1 species CUPRAC M1 M2 C AC
Fig. 1 Cu2+ ion reducing power (CUPRAC) assay of the extracts and curcumin.
terms of phenolic compounds were Ac M1 and M2. Studies on phenolic compounds have shown that these compounds are quite good antioxidant chemicals. Therefore, extracts rich in phenolic compounds have been shown good antioxidant activ-ity. Specifically, it was determined that M1 and M2 extracts which rich in phenolic compounds showed good antioxidant properties. In anticholinesterase activities; inhibition against the AChE enzyme was determined only for the extract of Ac and M1 of the O. hypericifolium, while the BChE enzyme was inhibited moderately by the all studied extracts. Therefore, it can be said that the extracts of these species having weak anticholinesterase effect while having a good antioxidant effect. This study supported that Origanum species are very important natural herbal products which are commonly used as an alternative to antioxidants in the pharmaceutical and food industry.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements
This work was supported by The Scientific and Technological Research Council of Turkey (TUBITAK) [grant number 113Z225].
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.org/10.1016/j.arabjc.2020.01.025.
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Table 8 Anticholinesterase activity of the the extracts.
AChE % Inhibition (200mg/mL) BChE % Inhibition (200mg/mL)
M1 C Ac M2 M1 C Ac M2 OB** 0 0 0 0 28.96 ± 0.65 46.69 ± 0.63 31.14 ± 0.58 0 OS** 0 0 0 0 31.46 ± 1.10 37.39 ± 1.20 59.42 ± 0.25 0 OSL** 0 0 0 0 3.00 ± 0.50 49.35 ± 0.82 48.76 ± 0.86 8.55 ± 0.40 OSP** 0 0 0 0 37.34 ± 0.37 35.59 ± 0.38 21.09 ± 0.21 0 OH** 51.32 ± 2.69 0 49.81 ± 0.53 0 54.91 ± 0.85 33.79 ± 0.86 62.08 ± 0.55 0 Galanthamine* 80.24 ± 0.28 82.10 ± 0.51 82.10 ± 0.51 80.24 ± 0.28 80.78 ± 1.22 82.05 ± 0.48 82.05 ± 0.48 80.78 ± 1.22 * Positive control. **
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